Bone morphogenetic protein (bmp)-17 and bmp-18 compositions

ABSTRACT

Purified BMP-17 and BMP-18 proteins and processes for producing them are disclosed. DNA molecules encoding the BMP-17 and BMP-18 proteins are also disclosed. The proteins may be used in the treatment of bone, cartilage, other connective tissue defects and disorders, including tendon, ligament and meniscus, in wound healing and related tissue repair, as well as for treatment of disorders and defects to tissues which include epidermis, nerve, muscle, including cardiac muscle, and other tissues and wounds, and organs such as liver, lung, epithelium, brain, spleen, cardiac, pancreas and kidney tissue. The proteins may also be useful for the induction of growth and/or differentiation of undifferentiated embryonic and stem cells.

[0001] The present invention relates to a novel family of purifiedproteins designated as Bone Morphogenetic Proteins (BMP)-17 and BMP-18,related proteins, DNA encoding them, and processes for obtaining them.These proteins may be used to induce bone and/or cartilage or otherconnective tissue formation, and in wound healing and tissue repair.These proteins may also be used for augmenting the activity of otherbone morphogenetic proteins.

BACKGROUND OF THE INVENTION

[0002] The search for the molecule or molecules responsible for thebone-, cartilage-, and other connective tissue-inductive activitypresent in bone and other tissue extracts has led to the discovery of anovel set of molecules called the Bone Morphogenetic Proteins (BMPs).The structures of several proteins, designated BMP-1 through BMP-16 havepreviously been elucidated. The unique inductive activities of theseproteins, along with their presence in bone, suggests that they areimportant regulators of bone repair processes, and may be involved inthe normal maintenance of bone tissue. There is a need to identifywhether additional proteins, particularly human proteins, exist whichplay a role in these processes. The present invention relates to theidentification of such a novel human protein, which the inventors havedesignated human BMP-17 and BMP-18.

[0003] Human BMP-17 and BMP-18 appear to be human homologs of a murineprotein called Lefty. The nucleotide and amino acid sequences of murineLefty are described in Zhou et al., Nature, 361:543-547 (1993). Themurine Lefty gene has been described as being expressed in the mousenode during gastrulation. A related human protein, designatedendometrial bleeding associated factor [EBAF] was published inKothapelli et al., J. Clin. Invest., 99:2342-2350 (1997).

SUMMARY OF THE INVENTION

[0004] As used herein, the term BMP-17 and BMP-18 proteins refer to thehuman BMP-17 and BMP-18 proteins, having the amino acid sequencesspecified in SEQUENCE (SEQ) ID NO:2 and SEQ ID NO: 4, as well as DNAsequences encoding the BMP-17 and BMP-18 proteins, such as the nativehuman sequences shown in SEQ ID NO:1 and SEQ ID NO: 3. Also included arenaturally occurring allelic sequences of SEQ ID NO:1 and 3, andequivalent degenerative codon sequences of the above.

[0005] The BMP-17 (SEQ ID NO: 1) and BMP-18 (SEQ ID NO: 3) DNA sequencesand amino acid sequences (SEQ ID NO: 2 and 4, respectively) are setforth in the Sequence Listings. BMP-17 and BMP-18 proteins may becapable of inducing the formation of cartilage, bone, or otherconnective tissue, or combinations thereof. The cartilage and/or boneand/or other connective tissue formation activity in the rat boneformation assay described below. BMP-17 and BMP-18 proteins may befurther characterized by the ability to demonstrate effects upon thegrowth and/or differentiation of embryonic cells and/or stem cells.Thus, the proteins or compositions of the present invention may also beuseful for treating cell populations, such as embryonic cells or stemcell populations, to enhance or enrich the growth and/or differentiationof the cells. Alternatively, the proteins or compositions of the presentinvention may also be useful for maintenance of a cell population,including differentiated cell populations, for example, neuronal cells,epithelial cells, dendritic cells, chondrocytes, osteocytes, musclecells or cells of other differentiated phenotypes.

[0006] Human BMP-17 proteins may be produced by culturing a celltransformed with a DNA sequence comprising nucleotide a DNA sequenceencoding the mature BMP-17 polypeptide, comprising nucleotide #427 tonucleotide #1098 as shown in SEQ ID NO: 1, and recovering and purifyingfrom the culture medium a protein characterized by the amino acidsequence comprising amino acids #1 to #224 as shown in SEQ ID NO:2substantially free from other proteinaceous materials with which it isco-produced. For production in mammalian cells, the DNA sequence furthercomprises a DNA sequence encoding a suitable propeptide 5′ to and linkedin frame to the nucleotide sequence encoding the mature BMP-17-relatedpolypeptide.

[0007] Human BMP-18 proteins may be produced by culturing a celltransformed with a DNA sequence comprising nucleotide a DNA sequenceencoding the mature BMP-18 polypeptide, comprising nucleotide #406 tonucleotide #1098 as shown in SEQ ID NO: 3, and recovering and purifyingfrom the culture medium a protein characterized by the amino acidsequence comprising amino acids #1 to #231 as shown in SEQ ID NO:4substantially free from other proteinaceous materials with which it isco-produced. For production in mammalian cells, the DNA sequence furthercomprises a DNA sequence encoding a suitable propeptide 5′ to and linkedin frame to the nucleotide sequence encoding the mature BMP-18-relatedpolypeptide.

[0008] The propeptide may be the native BMP-17 or BMP-18-relatedpropeptide, or may be a propeptide from another protein of the TGF-βsuperfamily. Where the native BMP-17 and BMP-18 propeptide is used,human BMP-17 and BMP-18 maybe produced by culturing a cell transformedwith a DNA sequence comprising a DNA sequence encoding the full BMP-17and BMP-18 polypeptide, comprising nucleotides #1 to #1098 as shown inSEQ ID NO: 1, or nucleotides #1 to #1098 as shown in SEQ ID NO: 3,producing a protein characterized by the amino acid sequence comprisingamino acids #-142 to #224 as shown in SEQ ID NO:2, of which amino acids#-142 to -1 comprise the native propeptide of human BMP-17; or #-135 to#231 of SEQ ID NO: 4 of which amino acids #-135 to -1 comprise thenative propeptide of human BMP-18, and recovering and purifying from theculture medium a protein characterized by the amino acid sequencecomprising amino acids #1 to #224 as shown in SEQ ID NO:2, or #1 to #231of SEQ ID NO:4, respectively, substantially free from otherproteinaceous materials with which it is co-produced.

[0009] Based in part upon the Von Heginje signal peptide predictionalgorithm, approximately the first 17 to 23 amino acids of SEQ ID NO: 2and 4 appear to be involved in signalling for the secretion of themature peptide. Accordingly, in one embodiment of the invention, DNAencoding a signal peptide, such as the native BMP-17 or BMP-18 signalpeptide, or another recognized signal peptide, may be linked directly tothe sequence encoding the mature BMP-17 or BMP-18 peptide.

[0010] It is expected that other species, particularly human, have DNAsequences homologous to human BMP-17 and BMP-18 protein. The invention,therefore, includes methods for obtaining the DNA sequences encodinghuman BMP-17 and BMP-18 proteins, the DNA sequences obtained by thosemethods, and the human proteins encoded by those DNA sequences. Thismethod entails utilizing the human BMP-17 and BMP-18 nucleotidesequences or portions thereof to design probes to screen libraries forthe corresponding gene from other species or coding sequences orfragments thereof from using standard techniques. Thus, the presentinvention may include DNA sequences from other species, which arehomologous to human BMP-17 and BMP-18 proteins and can be obtained usingthe human BMP-17 and/or BMP-18 sequences. The present invention may alsoinclude functional fragments of the human BMP-17 and BMP-18 proteins,and DNA sequences encoding such functional fragments, as well asfunctional fragments of other related proteins. The ability of such afragment to function is determinable by assay of the protein in thebiological assays described for the assay of the BMP-17 and BMP-18proteins. DNA sequences encoding the complete mature human BMP-17 (SEQID NO: 1 and BMP-18 protein (SEQ ID NO:3) and the corresponding aminoacid sequences (SEQ ID NO:2 and 4, respectively) are set forth herein.The BMP-17 and BMP-18 proteins of the present invention, such as humanBMP-17 and BMP-18, may be produced by culturing a cell transformed withthe correlating DNA sequence, such as the human BMP-17 and BMP-18 DNAsequence, and recovering and purifying protein, such as BMP-17 orBMP-18, from the culture medium. The purified expressed protein issubstantially free from other proteinaceous materials with which it isco-produced, as well as from other contaminants. The recovered purifiedprotein is contemplated to exhibit cartilage and/or bone and/orconnective tissue formation activity. Thus, the proteins of theinvention may be further characterized by the ability to demonstratecartilage and/or bone and/or other connective tissue formation activityin the rat bone formation assay described below. BMP-17 and BMP-18proteins may be further characterized by the ability to demonstrateeffects upon the growth and/or differentiation of embryonic cells and/orstem cells. Thus, the proteins or compositions of the present inventionmay also be characterized by their ability to enhance or enrich thegrowth and/or differentiation of the cells.

[0011] Another aspect of the invention provides pharmaceuticalcompositions containing a therapeutically effective amount of humanBMP-17 and/or BMP-18 protein, in a pharmaceutically acceptable vehicleor carrier. These compositions of the invention may be used in theformation of bone. These compositions may further be utilized for theformation of cartilage, or other connective tissue, including tendon,ligament, meniscus and other connective tissue, as well as combinationsof the above, for example regeneration of the tendon-to-bone attachmentapparatus. The compositions of the present invention, such ascompositions of human BMP-17 and/or BMP-18, may also be used for woundhealing and tissue repair. Compositions of the invention may furtherinclude at least one other therapeutically useful agent such as the BMPproteins BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosedfor instance in U.S. Pat. Nos. 5,108,922; 5,013,649; 5,116,738;5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publicationWO91/18098; and BMP-9, disclosed in PCT publication WO93/00432, BMP-10,disclosed in U.S. Pat. No. 5,637,480; BMP-11, disclosed in U.S. Pat. No.5,639,638, or BMP-12 or BMP-13, disclosed in U.S. Pat. No. 5,658,882,BMP-15, disclosed U.S. Pat. No. 5,635,372 and BMP-16, disclosed inco-pending patent application Ser. No. 08/715,202. Other compositionswhich may also be useful include Vgr-2, and any of the growth anddifferentiation factors [GDFs], including those described in PCTapplications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681;WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others. Also usefulin the present invention may be BIP, disclosed in WO94/01557; HP00269,disclosed in JP Publication number: 7-250688; and MP52, disclosed in PCTapplication WO93/16099. The disclosures of all of the above applicationsare hereby incorporated by reference.

[0012] The compositions of the invention may comprise, in addition to aBMP-17 and/or -18-related protein, other therapeutically useful agentsincluding growth factors such as epidermal growth factor (EGF),fibroblast growth factor (FGF), transforming growth factor (TGF-α andTGF-β), activins, inhibins, and insulin-like growth factor (IGF). Thecompositions may also include an appropriate matrix for instance, forsupporting the composition and providing a surface for bone and/orcartilage and/or other connective tissue growth. The matrix may provideslow release of the osteoinductive protein and/or the appropriateenvironment for presentation thereof.

[0013] The BMP-17 and/or BMP-18 containing compositions may be employedin methods for treating a number of bone and/or cartilage and/or otherconnective tissue defects, periodontal disease and healing of varioustypes of tissues and wounds. The tissue and wounds which may be treatedinclude epidermis, nerve, muscle, including cardiac muscle, and othertissues and wounds, and other organs such as liver, lung, epithelium,brain, spleen, cardiac, pancreas and kidney tissue. These methods,according to the invention, entail administering to a patient needingsuch bone and/or cartilage and/or other connective tissue formation,wound healing or tissue repair, an effective amount of a BMP-17 and/orBMP-18 protein. The BMP-17 and/or BMP-18 containing compositions mayalso be used to treat or prevent such conditions as osteoarthritis,osteoporosis, and other abnormalities of bone, cartilage, muscle,tendon, ligament or other connective tissue, organs such as liver, lung,epithelium, brain, spleen, cardiac, pancreas and kidney tissue, andother tissues. These methods may also entail the administration of aprotein of the invention in conjunction with at least one other BMPprotein as described above. In addition, these methods may also includethe administration of a BMP-17 and/or BMP-18 protein with other growthfactors including EGF, FGF, TGF-α, TGF-β, activin, inhibin and IGF.

[0014] Still a further aspect of the invention are DNA sequences codingfor expression of a BMP-17 and/or BMP-18 protein. Such sequences includethe sequence of nucleotides in a 5′ to 3′ direction illustrated in SEQID NO: 1 or SEQ ID NO: 3, DNA sequences which, but for the degeneracy ofthe genetic code, are identical to the DNA sequence SEQ ID NO: 1 or SEQID NO: 3, and encode the protein of SEQ ID NO: 2 or SEQ ID NO: 4.Further included in the present invention are DNA sequences whichhybridize under stringent conditions with the DNA sequence of SEQ ID NO:1 or SEQ ID NO: 3 and encode a protein having the ability to induceformation of cartilage, bone and/or other connective tissue, organs suchas liver, lung, epithelium, brain, spleen, cardiac, pancreas and kidneytissue, or other activities disclosed for BMP-17 and BMP-18. PreferredDNA sequences include those which hybridize under stringent conditions[For example, see conditions described in Maniatis et al, MolecularCloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982),pages 387 to 389]. It is generally preferred that such DNA sequencesencode a polypeptide which is at least about 80% homologous, and morepreferably at least about 90% homologous, to the conserved C-terminalcysteine structure of the amino acid sequence shown in SEQ ID NO:2 orSEQ ID NO: 4. Finally, allelic or other variations of the sequences ofSEQ ID NO: 1 or SEQ ID NO: 3, whether or not such nucleotide changesresult in changes in the peptide sequence, where the peptide sequenceretains one or more BMP-17 and/or BMP-18 activity, are also included inthe present invention. The present invention also includes fragments ofthe DNA sequence of BMP-17 and/or BMP-18 shown in SEQ ID NO: 1 or SEQ IDNO: 3 which encode a polypeptide which retains the activity of BMP-17and/or BMP-18 protein.

[0015] The DNA sequences of the present invention are useful, forexample, as probes for the detection of mRNA encoding BMP-17 and/orBMP-18 in a given cell population. Thus, the present invention includesmethods of detecting or diagnosing genetic disorders involving theBMP-17 and/or BMP-18 gene, or disorders involving cellular, organ ortissue disorders in which BMP-17 and/or BMP-18 is irregularlytranscribed or expressed. The DNA sequences may also be useful forpreparing vectors for gene therapy applications as described below.

[0016] A further aspect of the invention includes vectors comprising aDNA sequence as described above in operative association with anexpression control sequence therefor. These vectors may be employed in anovel process for producing a BMP-17 and/or BMP-18 protein of theinvention in which a cell line transformed with a DNA sequence encodinga BMP-17 and/or BMP-18 protein in operative association with anexpression control sequence therefor, is cultured in a suitable culturemedium and a BMP-17 and/or BMP-18-related protein is recovered andpurified therefrom. This process may employ a number of known cells bothprokaryotic and eukaryotic as host cells for expression of thepolypeptide. The vectors may be used in gene therapy applications. Insuch use, the vectors may be transfected into the cells of a patient exvivo, and the cells may be reintroduced into a patient. Alternatively,the vectors may be introduced into a patient in vivo through targetedtransfection.

[0017] Still a further aspect of the invention are BMP-17 and/or BMP-18proteins or polypeptides. Such polypeptides are characterized by havingan amino acid sequence including the sequence illustrated in SEQ ID NO:2 or SEQ ID NO: 4, variants of the amino acid sequence of SEQ ID NO: 2or SEQ ID NO: 4, including naturally occurring allelic variants, andother variants in which the protein retains the ability to induce theformation of cartilage and/or bone and/or other connective tissue, orother organs such as liver, lung, epithelium, brain, spleen, cardiac,pancreas and kidney tissue, or other activity characteristic of BMP-17and/or BMP-18. Preferred polypeptides include a polypeptide which is atleast about 80% homologous, and more preferably at least about 90%homologous, to the mature human BMP-17 and/or BMP-18 amino acid sequenceshown in SEQ ID NO:2 or SEQ ID NO: 4. Finally, allelic or othervariations of the sequences of SEQ ID NO: 2 or SEQ ID NO: 4, whethersuch amino acid changes are induced by mutagenesis, chemical alteration,or by alteration of DNA sequence used to produce the polypeptide, wherethe peptide sequence still has BMP-17 and/or BMP-18 activity, are alsoincluded in the present invention. The present invention also includesfragments of the amino acid sequence of BMP-17 and/or BMP-18 shown inSEQ ID NO: 2 or SEQ ID NO: 4 which retain the activity of BMP-17 and/orBMP-18 protein.

[0018] The purified proteins of the present inventions may be used togenerate antibodies, either monoclonal or polyclonal, to human BMP-17and/or BMP-18 and/or other BMP-17 and/or BMP-18-related proteins, usingmethods that are known in the art of antibody production. Thus, thepresent invention also includes antibodies to human BMP-17 and/or BMP-18and/or other related proteins. The antibodies may be useful forpurification of BMP-17 and/or BMP-18 and/or other BMP-17 and/or BMP-18related proteins, or for inhibiting or preventing the effects of BMP-17and/or BMP-18 related proteins. The BMP-17 and/or BMP-18 protein andrelated proteins may be useful for inducing the growth and/ordifferentiation of embryonic cells and/or stem cells. Thus, the proteinsor compositions of the present invention may also be useful for treatingrelatively undifferentiated cell populations, such as embryonic cells orstem cell populations, to enhance or enrich the growth and/ordifferentiation of the cells. The treated cell populations may be usefulfor implantation and for gene therapy applications.

[0019] Description of the Sequences

[0020] SEQ ID NO:1 is a nucleotide sequence containing nucleotidesequence encoding the entire mature human BMP-17 polypeptide.

[0021] SEQ ID NO:2 is a amino acid sequence containing the mature humanBMP-17 polypeptide sequence.

[0022] SEQ ID NO: 3 is a nucleotide sequence containing nucleotidesequence encoding the entire mature human BMP-18 polypeptide.

[0023] SEQ ID NO: 4 is a amino acid sequence containing the mature humanBMP-17 polypeptide sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The human BMP-17 and BMP-18 sequences of the present inventionmay be obtained using the whole or fragments of the murine Lefty DNAsequence, or a partial human BMP-17 or BMP-18 sequence, as a probe.Thus, the human BMP-17 and BMP-18 DNA sequence comprise the DNA sequenceof nucleotides #1 to #1098 of SEQ ID NO: 1 or #1 to #1098 of SEQ IDNO:3. The human BMP-17 and BMP-18 proteins comprise the sequences ofamino acids #-142 to #224 of SEQ ID NO: 2, or #-135 to #231 of SEQ IDNO: 4, respectively. The mature human BMP-17 and BMP-18 proteins areencoded by nucleotides #427 to #1098 of SEQ ID NO:1 and #406 to #1098 ofSEQ ID NO:3, respectively, and comprises the sequence of amino acids #1to #224 of SEQ ID NO:2, or #1 to #231 of SEQ ID NO: 4, respectively.

[0025] It is expected that human BMP-17 and BMP-18 polypeptides, asexpressed by mammalian cells such as CHO cells, exists as aheterogeneous population of active species of BMP-17 and BMP-18 proteinswith varying N-termini. It is expected that active species will comprisean amino acid sequence beginning with the residue at amino acid #109 or#124 of SEQ ID NO:2 or #116 or #131 of SEQ ID NO: 4, respectively, orwill comprise additional amino acid sequence further in the N-terminaldirection. Thus, it is expected that DNA sequences encoding activeBMP-17 and BMP-18 polypeptides will comprise a nucleotide sequencecomprising nucleotides #1, #232, #406, #427, #751 or #796 to #1059 or#1098 of SEQ ID NO: 1, or #1, #232, #406, #427, #751 or #796 to #1059 or#1098 of SEQ ID NO: 3, respectively. Accordingly, active species ofhuman BMP-17 and BMP-18 are expected to include those comprising aminoacids #-142, #-65, #-7, #1, #109 or #124 to #211 or #224 of SEQ ID NO:2,or #-135, #-58, #1, #8, #116 or #131 to #218 or #231 of SEQ ID NO: 4,respectively.

[0026] A host cell may be transformed with a coding sequence encoding apropeptide suitable for the secretion of proteins by the host cell islinked in proper reading frame to the coding sequence for the matureBMP-17 and BMP-18 protein. For example, see U.S. Pat. No. 5,658,882, inwhich the propeptide of BMP-2 is fused to the DNA encoding a matureBMP-12 protein. The disclosure of this reference is hereby incorporatedby reference. Thus, the present invention includes chimeric DNAmolecules comprising a DNA sequence encoding a propeptide from a memberof the TGF-β superfamily of proteins, other than BMP-17 and BMP-18, islinked in correct reading frame to a DNA sequence encoding human BMP-17or BMP-18 protein, or a related protein. The term “chimeric” is used tosignify that the propeptide originates from a different polypeptide thanthe native BMP-17 or BMP-18 protein.

[0027] The N-terminus of one active species of human BMP-17 is expectedto be experimentally determined by expression in E. coli to be asfollows: [M]ARVTV. Thus, it appears that the N-terminus of this speciesof BMP-17 is at amino acid #1 of SEQ ID NO: 1, and a DNA sequenceencoding said species of BMP-17 would comprise nucleotides #427 to #1098of SEQ ID NO: 1. The apparent molecular weight of human BMP-17 monomeris expected to be experimentally determined by SDS-PAGE to beapproximately 24.8 kD on a Novex 16% tricine gel. The human BMP-17protein is expected to exist as a clear, colorless solution in 0.1%trifluoroacetic acid.

[0028] It is expected that other BMP-17 proteins, as expressed bymammalian cells such as CHO cells, also exist as a heterogeneouspopulation of active species of BMP-17-related protein with varyingN-termini. For example, it is expected that active species of humanBMP-17 protein will comprise an amino acid sequence beginning with thecysteine residue at amino acid #109 or the glutamic acid residue atposition #124 of SEQ ID NO 2 or will comprise additional amino acidsequence further in the N-terminal direction. Thus, it is expected thatDNA sequences encoding active BMP-17 proteins include those whichcomprise a nucleotide sequence comprising nucleotides #1, 232, 406, 427,751 or 796 to #1059 or 1098 of SEQ ID NO: 1. Accordingly, active humanBMP-17 proteins include those comprising amino acids #-142, -65, -7, 1,109 or 124 to #211 or 224 of SEQ ID NO: 2.

[0029] The N-terminus of one active species of human BMP-18 is expectedto be experimentally determined by expression in E. coli to be asfollows: [M]LSPRS. Thus, it appears that the N-terminus of this speciesof BMP-18 is at amino acid #1 of SEQ ID NO: 3, and a DNA sequenceencoding said species of BMP-18 would comprise nucleotides #406 to #1098of SEQ ID NO:3. The apparent molecular weight of human BMP-18 monomer isexpected to be experimentally determined by SDS-PAGE to be approximately25.6 kD on a Novex 16% tricine gel. The human BMP-18 protein is expectedto exist as a clear, colorless solution in 0.1% trifluoroacetic acid.

[0030] It is expected that other BMP-18 proteins, as expressed bymammalian cells such as CHO cells, also exist as a heterogeneouspopulation of active species of BMP-18-related protein with varyingN-termini. For example, it is expected that active species of humanBMP-BMP-18 protein will comprise an amino acid sequence beginning withthe cysteine residue at amino acid #116 or the glutamic acid residue atposition #131 of SEQ ID NO:4 or will comprise additional amino acidsequence further in the N-terminal direction. Thus, it is expected thatDNA sequences encoding active BMP-18 proteins include those whichcomprise a nucleotide sequence comprising nucleotides #1, 232, 406, 427,751 or 796 to #1059 or 1098 of SEQ ID NO:3. Accordingly, active humanBMP-18 proteins include those comprising amino acids #-135, -58, 1, 8,116 or 131 to #218 or 231 of SEQ ID NO: 4.

[0031] The BMP-17 or BMP-18 proteins of the present invention, includepolypeptides having a molecular weight of about 24.8 to 25.6 kD inmonomeric form, said polypeptide comprising the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 4, respectively, and having the ability toinduce the formation of cartilage and/or bone and/or other connectivetissue in the Rosen-Modified Sampath-Reddi ectopic implant assay,described in the examples.

[0032] The BMP-17 or BMP-18 proteins recovered from the culture mediumare purified by isolating them from other proteinaceous materials fromwhich they are co-produced and from other contaminants present. BMP-17or BMP-18 proteins may be characterized by the ability to induce theformation of cartilage and/or bone and/or other connective tissue andother tissue repair and differentiation, for example, in the rat boneformation assay described below. In addition, BMP-17 or BMP-18 proteinsmay be further characterized by their effects upon the growth and/ordifferentiation of embryonic cells and/or stem cells. Thus, the proteinsor compositions of the present invention may be characterized by theembryonic stem cell assay described below.

[0033] The BMP-17 or BMP-18 proteins provided herein also includefactors encoded by the sequences similar to those of SEQ ID NO: 1 or SEQID NO: 3, but into which modifications or deletions are naturallyprovided (e.g. allelic variations in the nucleotide sequence which mayresult in amino acid changes in the polypeptide) or deliberatelyengineered. For example, synthetic polypeptides may wholly or partiallyduplicate continuous sequences of the amino acid residues of SEQ ID NO:2or SEQ ID NO: 4. These sequences, by virtue of sharing primary,secondary, or tertiary structural and conformational characteristicswith bone growth factor polypeptides of SEQ ID NO: 2 or SEQ ID NO: 4 maypossess biological properties in common therewith. It is know, forexample that numerous conservative amino acid substitutions are possiblewithout significantly modifying the structure and conformation of aprotein, thus maintaining the biological properties as well. Forexample, it is recognized that conservative amino acid substitutions maybe made among amino acids with basic side chains, such as lysine (Lys orK), arginine (Arg or R) and histidine (His or H); amino acids withacidic side chains, such as aspartic acid (Asp or D) and glutamic acid(Glu or E); amino acids with uncharged polar side chains, such asasparagine (Asn or N), glutamine (Gln or Q), serine (Ser or S),threonine (Thr or T), and tyrosine (Tyr or Y); and amino acids withnonpolar side chains, such as alanine (Ala or A), glycine (Gly or G),valine (Val or V), leucine (Leu or L), isoleucine (ile or I), proline(Pro or P), phenylalanine (Phe or F), methionine (Met or M), tryptophan(Trp or W) and cysteine (Cys or C). Thus, these modifications anddeletions of the native BMP-17 or BMP-18 may be employed as biologicallyactive substitutes for naturally-occurring BMP-17 or BMP-18 and otherpolypeptides in therapeutic processes. It can be readily determinedwhether a given variant of BMP-17 or BMP-18 maintains the biologicalactivity of BMP-17 or BMP-18 by subjecting both BMP-17 or BMP-18 and thevariant of BMP-17 or BMP-18 to the assays described in the examples.

[0034] Other specific mutations of the sequences of BMP-17 or BMP-18proteins described herein involve modifications of glycosylation sites.These modifications may involve O-linked or N-linked glycosylationsites. For instance, the absence of glycosylation or only partialglycosylation results from amino acid substitution or deletion atasparagine-linked glycosylation recognition sites. The asparagine-linkedglycosylation recognition sites comprise tripeptide sequences which arespecifically recognized by appropriate cellular glycosylation enzymes.These tripeptide sequences are either asparagine-X-threonine orasparagine-X-serine, where X is usually any amino acid. A variety ofamino acid substitutions or deletions at one or both of the first orthird amino acid positions of a glycosylation recognition site (and/oramino acid deletion at the second position) results in non-glycosylationat the modified tripeptide sequence. Additionally, bacterial expressionof BMP-17 and BMP-18-related protein will also result in production of anon-glycosylated protein, even if the glycosylation sites are leftunmodified.

[0035] The present invention also encompasses the novel DNA sequences,free of association with DNA sequences encoding other proteinaceousmaterials, and coding for expression of BMP-17 and BMP-18 proteins.These DNA sequences include those depicted in SEQ ID NO: 1 or SEQ ID NO:3 in a 5′ to 3′ direction and those sequences which hybridize theretounder stringent hybridization washing conditions [for example, 0.1×SSC,0.1% SDS at 65° C.; see, T. Maniatis et al, Molecular Cloning (ALaboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to389] and encode a protein maintaining one or more of the activitiesdisclosed herein for BMP-17 or BMP-18. These DNA sequences also includethose which comprise the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3and those which hybridize thereto under stringent hybridizationconditions and encode a protein which maintain the other activitiesdisclosed for BMP-17 or BMP-18.

[0036] Similarly, DNA sequences which code for BMP-17 or BMP-18polypeptides coded for by the sequences of SEQ ID NO: 1 or SEQ ID NO: 3,respectively, or BMP-17 or BMP-18 polypeptides which comprise the aminoacid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, but which differ in codonsequence due to the degeneracies of the genetic code or allelicvariations (naturally-occurring base changes in the species populationwhich may or may not result in an amino acid change) also encode thenovel factors described herein. Variations in the DNA sequences of SEQID NO: 1 or SEQ ID NO: 3 which are caused by point mutations or byinduced modifications (including insertion, deletion, and substitution)to enhance the activity, half-life or production of the polypeptidesencoded are also encompassed in the invention.

[0037] Another aspect of the present invention provides a novel methodfor producing BMP-17 or BMP-18 polypeptides. The method of the presentinvention involves culturing a suitable cell line, which has beentransformed with a DNA sequence encoding a BMP-17 or BMP-18 polypeptideof the invention, under the control of known regulatory sequences. Thetransformed host cells are cultured and the BMP-17 or BMP-18polypeptides recovered and purified from the culture medium. Thepurified polypeptides are substantially free from other proteins withwhich they are co-produced as well as from other contaminants.

[0038] Suitable cells or cell lines may be mammalian cells, such asChinese hamster ovary cells (CHO). The selection of suitable mammalianhost cells and methods for transformation, culture, amplification,screening, product production and purification are known in the art.See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981), oralternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1650 (1985) orHowley et al, U.S. Pat. No. 4,419,446. Another suitable mammalian cellline, which is described in the accompanying examples, is the monkeyCOS-1 cell line. The mammalian cell CV-1 may also be suitable.

[0039] Bacterial cells may also be suitable hosts. For example, thevarious strains of E. coli (e.g., HB101, MC1061) are well-known as hostcells in the field of biotechnology. Various strains of B. subtilis,Pseudomonas, other bacilli and the like may also be employed in thismethod. For expression of the protein in bacterial cells, DNA encodingthe propeptide of BMP-17 or BMP-18 is generally not necessary.

[0040] Many strains of yeast cells known to those skilled in the art mayalso be available as host cells for expression of the polypeptides ofthe present invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

[0041] Another aspect of the present invention provides vectors for usein the method of expression of these novel BMP-17 or BMP-18polypeptides. Preferably the vectors contain the full novel DNAsequences described above which encode the novel factors of theinvention. Additionally, the vectors contain appropriate expressioncontrol sequences permitting expression of the BMP-17 or BMP-18polypeptide sequences. Alternatively, vectors incorporating modifiedsequences as described above are also embodiments of the presentinvention. Additionally, the sequence of SEQ ID NO:1 or SEQ ID NO: 3 orother sequences encoding BMP-17 or BMP-18 polypeptides could bemanipulated to express a mature BMP-17 or BMP-18 polypeptide by deletingBMP-17 or BMP-18 propeptide sequences and replacing them with sequencesencoding the complete propeptides of other BMP proteins or members ofthe TGF-β superfamily. Thus, the present invention includes chimeric DNAmolecules encoding a propeptide from a member of the TGF-β superfamilylinked in correct reading frame to a DNA sequence encoding a BMP-17 orBMP-18 polypeptide.

[0042] The vectors may be employed in the method of transforming celllines and contain selected regulatory sequences in operative associationwith the DNA coding sequences of the invention which are capable ofdirecting the replication and expression thereof in selected host cells.Regulatory sequences for such vectors are known to those skilled in theart and may be selected depending upon the host cells. Such selection isroutine and does not form part of the present invention.

[0043] A protein of the present invention, which induces cartilageand/or bone and/or other connective tissue formation in circumstanceswhere such tissue is not normally formed, has application in the healingof bone fractures and cartilage or other connective tissue defects inhumans and other animals. Such a preparation employing a BMP-17 orBMP-18 polypeptide may have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. A BMP-17 or BMP-18-related polypeptide may be used inthe treatment of periodontal disease, and in other tooth repairprocesses. Such agents may provide an environment to attractbone-forming cells, stimulate growth of bone-forming cells or inducedifferentiation of progenitors of bone-forming cells, and may alsosupport the regeneration of the periodontal ligament and attachmentapparatus, which connects bone and teeth. BMP-17 or BMP-18 polypeptidesof the invention may also be useful in the treatment of osteoporosis. Avariety of osteogenic, cartilage-inducing and bone inducing factors havebeen described. See, e.g., European patent applications 148,155 and169,016 for discussions thereof.

[0044] The proteins of the invention may also be used in wound healingand related tissue repair. The types of wounds include, but are notlimited to burns, incisions and ulcers. (See, e.g. PCT PublicationWO84/01106 for discussion of wound healing and related tissue repair).It is further contemplated that proteins of the invention may increaseneuronal, astrocytic and glial cell survival and therefore be useful intransplantation and treatment of conditions exhibiting a decrease inneuronal survival and repair. The proteins of the invention may furtherbe useful for the treatment of conditions related to other types oftissue, such as nerve, epidermis, muscle, and other organs such asliver, lung, epithelium, brain, spleen, cardiac, pancreas and kidneytissue. The proteins of the present invention may further be useful forthe treatment of relatively undifferentiated cell populations, such asembryonic cells, or stem cells, to enhance growth and/or differentiationof the cells. The proteins of the present invention may also have valueas a dietary supplement, or as a component of cell culture media. Forthis use, the proteins may be used in intact form, or may be predigestedto provide a more readily absorbed supplement.

[0045] The proteins of the invention may also have other usefulproperties characteristic of the TGF-β superfamily of proteins. Suchproperties include angiogenic, chemotactic and/or chemoattractantproperties, and effects on cells including induction of collagensynthesis, fibrosis, differentiation responses, cell proliferativeresponses and responses involving cell adhesion, migration andextracellular matrices. These properties make the proteins of theinvention potential agents for wound healing, reduction of fibrosis andreduction of scar tissue formation.

[0046] When dimerized as a homodimer or as a heterodimer with otherBMPs, with other members of the TGF-β superfamily of proteins, or withinhibin-α proteins or inhibin-β proteins, the BMP-17 or BMP-18heterodimer is expected to demonstrate effects on the production offollicle stimulating hormone (FSH), as described further herein. It isrecognized that FSH stimulates the development of ova in mammalianovaries (Ross et al., in Textbook of Endocrinology, ed. Williams, p. 355(1981) and that excessive stimulation of the ovaries with FSH will leadto multiple ovulations. FSH is also important in testicular function.Thus, BMP-17 or BMP-18 may be useful as a contraceptive based on theability of inhibins to decrease fertility in female mammals and decreasespermatogenesis in male mammals. Administration of sufficient amounts ofother inhibins can induce infertility in mammals. BMP-17 or BMP-18 mayalso be useful as a fertility inducing therapeutic, based upon theability of activin molecules in stimulating FSH release from cells ofthe anterior pituitary. See, for example, U.S. Pat. No. 4,798,885.BMP-17 or BMP-18 may also be useful for advancement of the onset offertility in sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as cows, sheep andpigs. It is further contemplated that BMP-17 or BMP-18 may be useful inmodulating hematopoiesis by inducing the differentiation of erythroidcells [see, e.g., Broxmeyer et al, Proc. Natl. Acad. Sci. USA,85:9052-9056 (1988) or Eto et al, Biochem. Biophys. Res. Comm.,142:1095-1103 (1987)], for suppressing the development of gonadal tumors[see, e.g., Matzuk et al., Nature, 360:313-319 (1992)] or for augmentingthe activity of bone morphogenetic proteins [see, e.g., Ogawa et al., J.Biol. Chem., 267:14233-14237 (1992)].

[0047] BMP-17 and BMP-18 proteins may be further characterized by theirability to modulate the release of follicle stimulating hormone (FSH) inestablished in vitro bioassays using rat anterior pituitary cells asdescribed [see, e.g., Vale et al, Endocrinology, 91:562-572 (1972); Linget al., Nature, 321:779-782 (1986) or Vale et al., Nature, 321:776-779(1986)]. It is contemplated that the BMP-17 or BMP-18 protein of theinvention, when composed as a heterodimer with inhibin α or inhibin βchains, will exhibit regulatory effects, either stimulatory orinhibitory, on the release of follicle stimulating hormone (FSH), fromanterior pituitary cells as described [Ling et al., Nature, 321:779-782(1986) or Vale et al., Nature, 321:776-779 (1986); Vale et al,Endocrinology, 91:562-572 (1972). Therefore, depending on the particularcomposition, it is expected that the BMP-17 or BMP-18 protein of theinvention may have contrasting and opposite effects on the release offollicle stimulating hormone (FSH) from the anterior pituitary.

[0048] Activin A (the homodimeric composition of inhibin β_(A)) has beenshown to have erythropoietic-stimulating activity [see e.g. Eto et al.,Biochem. Biophys. Res. Commun., 142:1095-1103 (1987) and Murata et al.,Proc. Natl. Acad. Sci. U.S.A., 85:2434-2438 (1988) and Yu et al.,Nature, 330:765-767 (1987)]. It is contemplated that the BMP-17 and -18proteins of the invention may have a similar erythropoietic-stimulatingactivity. This activity of the BMP-17 and BMP-18 proteins may be furthercharacterized by the ability of the BMP-17 and BMP-18 proteins todemonstrate erythropoietin activity in the biological assay performedusing the human K-562 cell line as described by [Lozzio et al., Blood,45:321-334 (1975) and U.S. Pat. No. 5,071,834].

[0049] A further aspect of the invention is a therapeutic method andcomposition for repairing fractures and other conditions related tocartilage and/or bone and/or other connective tissue defects orperiodontal diseases. The invention further comprises therapeuticmethods and compositions for wound healing and tissue repair. Suchcompositions comprise a therapeutically effective amount of at least oneof the BMP-17 or BMP-18-related proteins of the invention in admixturewith a pharmaceutically acceptable vehicle, carrier or matrix. It isfurther contemplated that compositions of the invention may increaseneuronal survival and therefore be useful in transplantation andtreatment of conditions exhibiting a decrease in neuronal survival.Compositions of the invention may further include at least one othertherapeutically useful agent, such as members of the TGF-β superfamilyof proteins, which includes the BMP proteins BMP-1, BMP-2, BMP-3, BMP-4,BMP-5, BMP-6 and BMP-7, disclosed for instance in U.S. Pat. Nos.5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905;BMP-8, disclosed in PCT publication WO91/18098; BMP-9, disclosed in U.S.Pat. No. 5,661,007; BMP-10, disclosed in U.S. Pat. No. 5,637,480;BMP-11, disclosed in U.S. Pat. No. 5,639,638; BMP-12 or BMP-13,disclosed in U.S. Pat. No. 5,658,882; or BMP-15, disclosed in U.S. Pat.No. 5,635,372; or BMP-16, disclosed in co-pending patent applicationSer. No. 08/715,202. Other compositions which may also be useful includeVgr-2, and any of the growth and differentiation factors [GDFs],including those described in PCT applications WO94/15965; WO94/15949;WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539; WO96/01845;WO96/02559 and others. Also useful in the present invention may be BIP,disclosed in WO94/01557; HP00269, disclosed in JP Publication number:7-250688; and MP52, disclosed in PCT application WO93/16099. Thedisclosures of the above applications are hereby incorporated byreference herein.

[0050] It is expected that human BMP-17 and BMP-18 protein may exist innature as monomers, or as homodimers or heterodimers. To promote theformation of dimers of BMP-17 or BMP-18 and useful proteins withincreased stability, one can genetically engineer the DNA sequence ofSEQ ID NO:1 or SEQ ID NO: 3 to provide one or more additional cysteineresidues to increase potential dimer formation. The resulting DNAsequence would be capable of producing a “cysteine added variant” ofBMP-17 or BMP-18. In a preferred embodiment, one would engineer the DNAsequence of SEQ ID NO: 1 or SEQ ID NO: 3 so that one or more codons maybe altered to a nucleotide triplet encoding a cysteine residue, such asTGT or TGC. Alternatively, one can produce “cysteine added variants” ofBMP-17 or BMP-18 protein by altering the sequence of the protein at theamino acid level by altering one or more amino acid residues of SEQ IDNO:2 or SEQ ID NO: 4 to cysteine. Production of “cysteine addedvariants” of proteins is described in U.S. Pat. No. 5,166,322, thedisclosure of which is hereby incorporated by reference. In preferredembodiments, the glutamic acid residue at position #173 of SEQ. ID NO: 2or position #180 of SEQ. ID NO: 4 is replaced by a cysteine residue.

[0051] It is expected that the proteins of the invention may act inconcert with or perhaps synergistically with other related proteins andgrowth factors. Further therapeutic methods and compositions of theinvention therefore comprise a therapeutic amount of at least one BMP-17or BMP-18 protein of the invention with a therapeutic amount of at leastone other member of the TGF-β superfamily of proteins, such as the BMPproteins disclosed in the applications described above. Suchcombinations may comprise separate molecules of the BMP proteins orheteromolecules comprised of different BMP moieties. For example, amethod and composition of the invention may comprise a disulfide linkeddimer comprising a BMP-17 or BMP-18 protein subunit and a subunit fromone of the “BMP” proteins described above. Thus, the present inventionincludes a purified BMP-17 or BMP-18-related polypeptide which is aheterodimer wherein one subunit comprises the amino acid sequence fromamino acid #1 to amino acid #224 of SEQ ID NO:2 or #1 to #231 of SEQ IDNO:4, and one subunit comprises an amino acid sequence for a bonemorphogenetic protein selected from the group consisting of BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-15, or BMP-16. A further embodiment may comprise aheterodimer of BMP-17 or BMP-18-related moieties, for example aheterodimer of human BMP-17 and human BMP-18 or of human BMP-17 and themurine Lefty protein, which is a homologue of human BMP-17 and BMP-18.Further, BMP-17 or BMP-18 protein may be combined with other agentsbeneficial to the treatment of the bone and/or cartilage and/or otherconnective tissue defect, wound, or tissue in question. These agentsinclude various growth factors such as epidermal growth factor (EGF),fibroblast growth factor (FGF), platelet derived growth factor (PDGF),transforming growth factors (TGF-αand TGF-β), activins, inhibins, andk-fibroblast growth factor (kFGF), parathyroid hormone (PTH),parathyroid hormone related peptide (PTHrP), leukemia inhibitory factor(LIB/HILA/DA), insulin-like growth factors (IGF-I and IGF-II). Portionsof these agents may also be used in compositions of the presentinvention. The preparation and formulation of such physiologicallyacceptable protein compositions, having due regard to pH, isotonicity,stability and the like, is within the skill of the art. The therapeuticcompositions are also presently valuable for veterinary applications dueto the lack of species specificity in BMP proteins. Particularlydomestic animals and thoroughbred horses in addition to humans aredesired patients for such treatment with the BMP-17 or BMP-18 proteinsof the present invention.

[0052] The therapeutic method includes administering the compositiontopically, systemically, or locally as an implant or device. Whenadministered, the therapeutic composition for use in this invention is,of course, in a pyrogen-free, physiologically acceptable form. Further,the composition may desirably be encapsulated or injected in a viscousform for delivery to the site of bone, cartilage or other connectivetissue or other tissue damage. Topical administration may be suitablefor wound healing and tissue repair. Therapeutically useful agents otherthan the BMP-17 or BMP-18 proteins which may also optionally be includedin the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with theBMP composition in the methods of the invention.

[0053] Preferably for bone and/or cartilage and/or other connectivetissue formation, the composition includes a matrix capable ofdelivering BMP-17 or BMP-18-related or other BMP proteins to the site ofbone and/or cartilage and/or other connective tissue damage, providing astructure for the developing bone and cartilage and other connectivetissue and optimally capable of being resorbed into the body. The matrixmay provide slow release of BMP-17 or BMP-18 protein and/or other boneinductive protein, as well as proper presentation and appropriateenvironment for cellular infiltration. Such matrices may be formed ofmaterials presently in use for other implanted medical applications.

[0054] The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the BMP-17 or BMP-18compositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined, such as sintered hydroxyapatite, bioglass, aluminates, or otherceramics. Matrices may be comprised of combinations of any of the abovementioned types of material, such as polylactic acid and hydroxyapatiteor collagen and tricalcium phosphate. The bioceramics may be altered incomposition, such as in calcium-aluminate-phosphate and processing toalter pore size, particle size, particle shape, and biodegradability.

[0055] The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the BMP-17 orBMP-18 protein, e.g. amount of bone or other tissue weight desired to beformed, the site of bone or tissue damage, the condition of the damagedbone tissue, the size of a wound, type of damaged tissue, the patient'sage, sex, and diet, the severity of any infection, time ofadministration and other clinical factors. The dosage may vary with thetype of matrix used in the reconstitution and the types of BMP proteinsin the composition. Generally, systemic or injectable administrationwill be initiated at a dose which is minimally effective, and the dosewill be increased over a preselected time course until a positive effectis observed. Subsequently, incremental increases in dosage will be madelimiting such incremental increases to such levels that produce acorresponding increase in effect, while taking into account any adverseaffects that may appear. The addition of other known growth factors,such as IGF I (insulin like growth factor I), to the final composition,may also effect the dosage.

[0056] Progress can be monitored by periodic assessment of bone ortissue growth and/or repair. The progress can be monitored, for example,x-rays, histomorphometric determinations and tetracycline labeling.

[0057] The following examples illustrate practice of the presentinvention with respect to human BMP-17 and human BMP-18 and other BMP-17and BMP-18-related proteins. The skilled artisan will recognize thatnumerous variations and modifications are possible. These variations andmodifications constitute part of the present invention.

EXAMPLES Example 1

[0058] Isolation of DNA

[0059] DNA sequences encoding human BMP-17 and BMP-18 and human BMP-17and BMP-18-related proteins may be isolated by various techniques knownto those skilled in the art using the sequence information provided inthe Sequence Listings.

[0060] Based on the knowledge of BMP proteins and other proteins withinthe TGF-β family, it is predicted that the carboxyl-terminal portion ofthese molecules (mature peptide) would exhibit greater sequenceconservation than the more amino-terminal portions (propeptide region).This sequence relationship between BMP proteins and other proteinswithin the TGF-β family enables those skilled in the art to design DNAprobes from the carboxyl-terminal encoding portion (mature peptideencoding region) of these molecules which can be utilized to identifyrelated BMP proteins and other proteins within the TGF-β family. Themature peptide encoding region of the murine lefty gene may be utilizedto identify human BMP-17 and BMP-18 and related proteins.

[0061] The bacteriophage AF02, which contains the DNA sequence encodinghuman BMP-17 has been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. ATCC under theaccession #202060 on Nov. 24, 1997. This deposit meets the requirementsof the Budapest Treaty of the International Recognition of the Depositof Microorganisms for the Purpose of Patent Procedure and Regulationsthereunder.

[0062] The bacteriophage AF04, which contains the DNA sequence encodinghuman BMP-18 has been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. ATCC under theaccession #20259 on Nov. 24, 1997. This deposit meets the requirementsof the Budapest Treaty of the International Recognition of the Depositof Microorganisms for the Purpose of Patent Procedure and Regulationsthereunder.

[0063] Based on the knowledge of other BMP proteins and other proteinswithin the TGF-β family, it is predicted that the human BMP-17 andBMP-18 precursor polypeptides would be cleaved at the multibasicsequence in agreement with a proposed consensus proteolytic processingsequence of Arg-X-X-Arg. Cleavage of the human BMP-17 precursorpolypeptide is expected to generate a 224 amino acid mature peptidebeginning with the amino acid alanine at position #1 of SEQ ID NO:2, ora 101 amino acid mature peptide beginning with the amino acid glutamicacid at position #124 of SEQ ID NO: 2. Cleavage of the human BMP-18precursor polypeptide is expected to generate a 231 amino acid maturepeptide beginning with the amino acid leucine at position #1 of SEQ IDNO:4, or a 101 amino acid mature peptide beginning with the amino acidglutamic acid at position #131 of SEQ ID NO: 4. The processing of humanBMP-17 or BMP-18 into the mature forms is expected to potentiallyinvolve dimerization and removal of the N-terminal region in a manneranalogous to the processing of the related protein TGF-β [Gentry et al.,Molec & Cell. Biol., 8:4162 (1988); Derynck et al. Nature, 316:701(1985)].

[0064] It is contemplated therefore that the mature active species ofhuman BMP-17 and BMP-18 may comprise homodimers of two polypeptidesubunits, each subunit comprising amino acids #1 to #224 of SEQ ID NO:2or #1 to #231 of SEQ ID NO:4, respectively, with a predicted molecularweight of approximately 49 to 52 kD. Further active species arecontemplated comprising at least amino acids #109 to #211 of SEQ ID NO:2or #116 to #218 of SEQ ID NO: 4, thereby including the first and lastconserved cysteine residues. As with other members of the TGF-β/BMPfamily of proteins, the carboxyl-terminal portion of the human BMP-17and BMP-18 polypeptides exhibit greater sequence conservation than themore amino-terminal portion.

[0065] The human BMP-17 (SEQ ID NO:1) or BMP-18 DNA sequence (SEQ IDNO:3), or a portion thereof, can be used as a probe to identify a humancell line or tissue which synthesizes human BMP-17 or BMP-18 or a humanBMP-17 or BMP-18-related mRNA. Briefly described, RNA is extracted froma selected cell or tissue source and either electrophoresed on aformaldehyde agarose gel and transferred to nitrocellulose, or reactedwith formaldehyde and spotted on nitrocellulose directly. Thenitrocellulose is then hybridized to a probe derived from the codingsequence of human BMP-17 and BMP-18.

[0066] Alternatively, the human BMP-17 or BMP-18 sequence may be used todesign oligonucleotide primers which will specifically amplify a portionof the human BMP-17 or BMP-18 or related encoding sequences. It iscontemplated that these human BMP-17 and human BMP-18 derived primerswould allow one to specifically amplify corresponding human BMP-17 orBMP-18 or related encoding sequences from mRNA, cDNA or genomic DNAtemplates. Once a positive source has been identified by one of theabove described methods, mRNA is selected by oligo (dT) cellulosechromatography and cDNA is synthesized and cloned in λgt10 or other λbacteriophage vectors known to those skilled in the art, for example,λZAP by established techniques (Toole et al., supra). It is alsopossible to perform the oligonucleotide primer directed amplificationreaction, described above, directly on a preestablished human cDNA orgenomic library which has been cloned into a λ bacteriophage vector. Insuch cases, a library which yields a specifically amplified DNA productencoding a portion of the human BMP-17 or BMP-18 or related proteincould be screened directly, utilizing the fragment of amplified humanBMP-17, BMP-18 or related encoding DNA as a probe.

[0067] Additional methods known to those skilled in the art may be usedto isolate other full-length cDNAs encoding human BMP-17 or BMP-18 orrelated proteins, or full length cDNA clones encoding BMP-17 or BMP-18or related proteins of the invention from species other than humans,particularly other mammalian species.

Example 2

[0068] W-20 BIOASSAYS

[0069] A. Description of W-20 cells

[0070] Use of the W-20 bone marrow stromal cells as an indicator cellline is based upon the conversion of these cells to osteoblast-likecells after treatment with a BMP protein [Thies et al, Journal of Boneand Mineral Research, 5:305 (1990); and Thies et al, Endocrinology,130:1318 (1992)]. Specifically, W-20 cells are a clonal bone marrowstromal cell line derived from adult mice by researchers in thelaboratory of Dr. D. Nathan, Children's Hospital, Boston, Mass.Treatment of W-20 cells with certain BMP proteins results in (1)increased alkaline phosphatase production, (2) induction of PTHstimulated cAMP, and (3) induction of osteocalcin synthesis by thecells. While (1) and (2) represent characteristics associated with theosteoblast phenotype, the ability to synthesize osteocalcin is aphenotypic property only displayed by mature osteoblasts. Furthermore,to date we have observed conversion of W-20 stromal cells toosteoblast-like cells only upon treatment with BMPs. In this manner, thein vitro activities displayed by BMP treated W-20 cells correlate withthe in vivo bone forming activity known for BMPs.

[0071] Below two in vitro assays useful in comparison of BMP activitiesof novel osteoinductive molecules are described.

[0072] B. W-20 Alkaline Phosphatase Assay Protocol

[0073] W-20 cells are plated into 96 well tissue culture plates at adensity of 10,000 cells per well in 200 μl of media (DME with 10% heatinactivated fetal calf serum, 2 mM glutamine and 100 Units/mlpenicillin+100 μg/ml streptomycin. The cells are allowed to attachovernight in a 95% air, 5% CO₂ incubator at 37° C. The 200 μl of mediais removed from each well with a multichannel pipettor and replaced withan equal volume of test sample delivered in DME with 10% heatinactivated fetal calf serum, 2 mM glutamine and 1%penicillin-streptomycin. Test substances are assayed in triplicate. Thetest samples and standards are allowed a 24 hour incubation period withthe W-20 indicator cells. After the 24 hours, plates are removed fromthe 37° C. incubator and the test media are removed from the cells. TheW-20 cell layers are washed 3 times with 200 μl per well ofcalcium/magnesium free phosphate buffered saline and these washes arediscarded. 50 μl of glass distilled water is added to each well and theassay plates are then placed on a dry ice/ethanol bath for quickfreezing. Once frozen, the assay plates are removed from the dryice/ethanol bath and thawed at 37° C. This step is repeated 2 more timesfor a total of 3 freeze-thaw procedures. Once complete, the membranebound alkaline phosphatase is available for measurement. 50 μl of assaymix (50 mM glycine, 0.05% Triton X-100, 4 mM MgCl₂, 5 mM p-nitrophenolphosphate, pH=10.3) is added to each assay well and the assay plates arethen incubated for 30 minutes at 37° C. in a shaking waterbath at 60oscillations per minute. At the end of the 30 minute incubation, thereaction is stopped by adding 100 μl of 0.2 N NaOH to each well andplacing the assay plates on ice. The spectrophotometric absorbance foreach well is read at a wavelength of 405 nanometers. These values arethen compared to known standards to give an estimate of the alkalinephosphatase activity in each sample. For example, using known amounts ofp-nitrophenol phosphate, absorbance values are generated. This is shownin Table I. TABLE I Absorbance Values for Known Standards ofP-Nitrophenol Phosphate P-nitrophenol phosphate umoles Mean absorbance(405 nm) 0.000 0 0.006 0.261 +/− .024 0.012 0.521 +/− .031 0.018 0.797+/− .063 0.024 1.074 +/− .061 0.030 1.305 +/− .083

[0074] Absorbance values for known amounts of BMPs can be determined andconverted to pmoles of p-nitrophenol phosphate cleaved per unit time asshown in Table II. TABLE II Alkaline Phosphatase Values for W-20 CellsTreating with BMP-2 BMP-2 concentration Absorbance Reading umolessubstrate ng/ml 405 nmeters per hour 0 0.645 0.024 1.56 0.696 0.026 3.120.765 0.029 6.25 0.923 0.036 12.50 1.121 0.044 25.0 1.457 0.058 50.01.662 0.067 100.0 1.977 0.080

[0075] These values are then used to compare the activities of knownamounts of BMP-17 and BMP-18 to BMP-2.

[0076] C. Osteocalcin RIA Protocol

[0077] W-20 cells are plated at 16cells per well in 24 well multiwelltissue culture dishes in 2 mls of DME containing 10% heat inactivatedfetal calf serum, 2 mM glutamine. The cells are allowed to attachovernight in an atmosphere of 95% air 5% CO₂ at 37° C. The next day themedium is changed to DME containing 10% fetal calf serum, 2 mM glutamineand the test substance in a total volume of 2 ml. Each test substance isadministered to triplicate wells. The test substances are incubated withthe W-20 cells for a total of 96 hours with replacement at 48 hours bythe same test medias. At the end of 96 hours, 50 μl of the test media isremoved from each well and assayed for osteocalcin production using aradiomimmunoassay for mouse osteocalcin. The details of the assay aredescribed in the kit manufactured by Biomedical Technologies Inc., 378Page Street, Stoughton, Mass. 02072. Reagents for the assay are found asproduct numbers BT-431 (mouse osteocalcin standard), BT-432 (Goatanti-mouse Osteocalcin), BT-431R (iodinated mouse osteocalcin), BT-415(normal goat serum) and BT-414 (donkey anti goat IgG). The RIA forosteocalcin synthesized by W-20 cells in response to BMP treatment iscarried out as described in the protocol provided by the manufacturer.

[0078] The values obtained for the test samples are compared to valuesfor known standards of mouse osteocalcin and to the amount ofosteocalcin produced by W-20 cells in response to challenge with knownamounts of BMP-2. The values for BMP-2 induced osteocalcin synthesis byW-20 cells is shown in Table III. TABLE III Osteocalcin Synthesis byW-20 Cells BMP-2 Concentration ng/ml Osteocalcin Synthesis ng/well  00.8  2 0.9  4 0.8  8 2.2  17 2.7  31 3.2  62 5.1 125 6.5 250 8.2 500 9.41000  10.0 

Example 3

[0079] Rosen Modified Sampath-Reddi Assay

[0080] A modified version of the rat bone formation assay described inSampath and Reddi, Proc. Natl. Acad. Sci. USA 80:6591-6595 (1983) isused to evaluate bone and/or cartilage and/or other connective tissueactivity of BMP proteins. This modified assay is herein called theRosen-modified Sampath-Reddi assay. The ethanol precipitation step ofthe Sampath-Reddi procedure is replaced by dialyzing (if the compositionis a solution) or diafiltering (if the composition is a suspension) thefraction to be assayed against water. The solution or suspension is thenequilibrated to 0.1% TFA. The resulting solution is added to 20 mg ofrat matrix. A mock rat matrix sample not treated with the protein servesas a control. This material is frozen and lyophilized and the resultingpowder enclosed in #5 gelatin capsules. The capsules are implantedsubcutaneously in the abdominal thoracic area of 21-49 day old male LongEvans rats. The implants are removed after 7-14 days. Half of eachimplant is used for alkaline phosphatase analysis [see, Reddi et a],Proc. Natl. Acad. Sci., 69:1601 (1972)].

[0081] The other half of each implant is fixed and processed forhistological analysis. 1 μm glcolmethacrylate sections are stained withVon Kossa and acid fuschin to score the amount of induced bone andcartilage and other connective tissue formation present in each implant.The terms +1 through +5 represent the area of each histological sectionof an implant occupied by new bone and/or cartilage cells and matrix. Ascore of +5 indicates that greater than 50% of the implant is new boneand/or cartilage produced as a direct result of protein in the implant Ascore of +4, +3, +2, and +1 would indicate that greater than 40%, 30%,20% and 10% respectively of the implant contains new cartilage and/orbone.

[0082] Alternatively, the implants are inspected for the appearance oftissue resembling embryonic tendon, which is easily recognized by thepresence of dense bundles of fibroblasts oriented in the same plane andpacked tightly together. [Tendon/ligament-like tissue is described, forexample, in Ham and Cormack, Histology (JB Lippincott Co. (1979), pp.367-369, the disclosure of which is hereby incorporated by reference].These findings may be reproduced in additional assays in whichtendon/ligament-like tissues are observed in the BMP-17 andBMP-18-related protein containing implants. The BMP-17 andBMP-18-related proteins of this invention may be assessed for activityon this assay.

Example 4

[0083] Expression of BMP-17 and BMP-18

[0084] In order to produce murine, human or other mammalian BMP-17 andBMP-18 proteins, the DNA encoding it is transferred into an appropriateexpression vector and introduced into mammalian cells or other preferredeukaryotic or prokaryotic hosts by conventional genetic engineeringtechniques. The preferred expression system for biologically activerecombinant human BMP-17 and BMP-18 is contemplated to be stablytransformed mammalian cells.

[0085] One skilled in the art can construct mammalian expression vectorsby employing the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or other DNAsequences encoding BMP-17 and BMP-18 or related proteins or othermodified sequences and known vectors, such as pCD [Okayama et al., Mol.Cell Biol., 2:161 (1982)], pJL3, pJL4 [Gough et al., EMBO J., 4:645-653(1985)] and pMT2 CXM. The mammalian expression vector pMT2 CXM is aderivative of p91023(b) (Wong et al., Science 228:810-815, 1985)differing from the latter in that it contains the ampicillin resistancegene in place of the tetracycline resistance gene and further contains aXhoI site for insertion of cDNA clones. The functional elements of pMT2CXM have been described (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci.USA 82:689-693) and include the adenovirus VA genes, the SV40 origin ofreplication including the 72 bp enhancer, the adenovirus major latepromoter including a 5′ splice site and the majority of the adenovirustripartite leader sequence present on adenovirus late mRNAs, a 3′ spliceacceptor site, a DHFR insert, the SV40 early polyadenylation site(SV40), and pBR322 sequences needed for propagation in E. coli.

[0086] Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF,which has been deposited with the American Type Culture Collection(ATCC), Rockville, Md. (USA) under accession number ATCC 67122. EcoRIdigestion excises the cDNA insert present in pMT2-VWF, yielding pMT2 inlinear form which can be ligated and used to transform E. coli HB 101 orDH-5 to ampicillin resistance. Plasmid pMT2 DNA can be prepared byconventional methods. pMT2 CXM is then constructed using loopout/inmutagenesis [Morinaga, et al., Biotechnology 84: 636 (1984). Thisremoves bases 1075 to 1145 relative to the Hind III site near the SV40origin of replication and enhancer sequences of pMT2. In addition itinserts the following sequence:

[0087] 5′ PO-CATGGGCAGCTCGAG-3′ at nucleotide 1145. This sequencecontains the recognition site for the restriction endonuclease Xho I. Aderivative of pMT2CXM, termed pMT23, contains recognition sites for therestriction endonucleases PstI, Eco RI, Sail and XhoI. Plasmid pMT2 CXMand pMT23 DNA may be prepared by conventional methods.

[0088] pEMC2β1 derived from pMT21 may also be suitable in practice ofthe invention. pMT21 is derived from pMT2 which is derived frompMT2-VWF. As described above EcoRI digestion excises the cDNA insertpresent in pMT-VWF, yielding pMT2 in linear form which can be ligatedand used to transform E. Coli HB 101 or DH-5 to ampicilln resistance.Plasmid pMT2 DNA can be prepared by conventional methods.

[0089] pMT21 is derived from pMT2 through the following twomodifications. First, 76 bp of the 5′ untranslated region of the DHFRcDNA including a stretch of 19 G residues from G/C tailing for cDNAcloning is deleted. In this process, a XhoI site is inserted to obtainthe following sequence immediately upstream from DHFR:5′-CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3′   PstI           Eco RI XhoI

[0090] Second, a unique ClaI site is introduced by digestion with EcoRVand XbaI, treatment with Klenow fragment of DNA polymerase I, andligation to a ClaI linker (CATCGATG). This deletes a 250 bp segment fromthe adenovirus associated RNA (VAI) region but does not interfere withVAI RNA gene expression or function. pMT21 is digested with EcoRI andXhoI, and used to derive the vector pEMC2B1.

[0091] A portion of the EMCV leader is obtained from pMT2-ECAT1 [S. K.Jung, et al, J. Virol 63:1651-1660 (1989)] by digestion with Eco RI andPstI, resulting in a 2752 bp fragment. This fragment is digested withTaqI yielding an Eco RI-TaqI fragment of 508 bp which is purified byelectrophoresis on low melting agarose gel. A 68 bp adapter and itscomplementary strand are synthesized with a 5′ TaqI protruding end and a3′ XhoI protruding end which has the following sequence:5′-CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT   TaqIGAAAAACACGATTGC-3′          XhoI

[0092] This sequence matches the EMC virus leader sequence fromnucleotide 763 to 827. It also changes the ATG at position 10 within theEMC virus leader to an ATT and is followed by a XhoI site. A three wayligation of the pMT21 Eco RI-16hoI fragment, the EMC virus EcoRI-TaqIfragment, and the 68 bp oligonucleotide adapter TaqI-16hoI adapterresulting in the vector pEMC2⊕1.

[0093] This vector contains the SV40 origin of replication and enhancer,the adenovirus major late promoter, a cDNA copy of the majority of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,DHFR and β-lactamase markers and an EMC sequence, in appropriaterelationships to direct the high level expression of the desired cDNA inmammalian cells.

[0094] The construction of vectors may involve modification of theBMP-17 and BMP-18-related DNA sequences. For instance, BMP-17 and BMP-18cDNA can be modified by removing the non-coding nucleotides on the 5′and 3′ ends of the coding region. The deleted non-coding nucleotides mayor may not be replaced by other sequences known to be beneficial forexpression. These vectors are transformed into appropriate host cellsfor expression of BMP-17 and BMP-18-related proteins. Additionally, thesequence of SEQ ID NO:1, SEQ ID NO: 3 or other sequences encoding BMP-17and BMP-18-related proteins can be manipulated to express a matureBMP-17 or BMP-18 or related protein by deleting BMP-17 or BMP-18encoding propeptide sequences and replacing them with sequences encodingthe complete propeptides of other BMP proteins.

[0095] One skilled in the art can manipulate the sequences of SEQ ID NO:1 or SEQ ID NO: 3 by eliminating or replacing the mammalian regulatorysequences flanking the coding sequence with bacterial sequences tocreate bacterial vectors for intracellular or extracellular expressionby bacterial cells. For example, the coding sequences could be furthermanipulated (e.g. ligated to other known linkers or modified by deletingnon-coding sequences therefrom or altering nucleotides therein by otherknown techniques). The modified BMP-17 or BMP-18-related coding sequencecould then be inserted into a known bacterial vector using proceduressuch as described in T. Taniguchi et al., Proc. Natl Acad. Sci. USA,77:5230-5233 (1980). This exemplary bacterial vector could then betransformed into bacterial host cells and a BMP-17 and BMP-18-relatedprotein expressed thereby. For a strategy for producing extracellularexpression of BMP-17 or BMP-18-related proteins in bacterial cells, see,e.g. European patent application EPA 177,343.

[0096] Similar manipulations can be performed for the construction of aninsect vector [See, e.g. procedures described in published Europeanpatent application 155,476] for expression in insect cells. A yeastvector could also be constructed employing yeast regulatory sequencesfor intracellular or extracellular expression of the factors of thepresent invention by yeast cells. [See, e.g., procedures described inpublished PCT application WO86/00639 and European patent application EPA123,289].

[0097] A method for producing high levels of a BMP-17 or BMP-18 orrelated protein of the invention in mammalian cells may involve theconstruction of cells containing multiple copies of the heterologousBMP-17 or BMP-18 gene. The heterologous gene is linked to an amplifiablemarker, e.g. the dihydrofolate reductase (DHFR) gene for which cellscontaining increased gene copies can be selected for propagation inincreasing concentrations of methotrexate (MTX) according to theprocedures of Kaufman and Sharp, J. Mol. Biol., 159:601-629 (1982). Thisapproach can be employed with a number of different cell types.

[0098] For example, a plasmid containing a DNA sequence for a BMP-17 orBMP-18 or related protein of the invention in operative association withother plasmid sequences enabling expression thereof and the DHFRexpression plasmid pAdA26SV(A)3 [Kaufman and Sharp, Mol. Cell. Biol.,2:1304 (1982)] can be co-introduced into DHFR-deficient CHO cells,DUKX-BII, by various methods including calcium phosphate coprecipitationand transfection, electroporation or protoplast fusion. DHFR expressingtransformants are selected for growth in alpha media with dialyzed fetalcalf serum, and subsequently selected for amplification by growth inincreasing concentrations of MTX (e.g. sequential steps in 0.02, 0.2,1.0 and 5 uM MTX) as described in Kaufman et al., Mol Cell Biol., 5:1650(1983). Transformants are cloned, and biologically active BMP-17 orBMP-18 expression is monitored by the Rosen-modified Sampath-Reddi ratbone formation assay described above in Example 3. BMP-17 and BMP-18protein expression should increase with increasing levels of MTXresistance. BMP-17 and BMP-18 polypeptides are characterized usingstandard techniques known in the art such as pulse labeling with [35S]methionine or cysteine and polyacrylamide gel electrophoresis. Similarprocedures can be followed to produce other related BMP-17 and BMP-18 orrelated proteins.

Example 5

[0099] Biological Activity of Expressed BMP-17 and BMP-18

[0100] To measure the biological activity of the expressed BMP-17 andBMP-18 or related proteins obtained in Example 4 above, the proteins arerecovered from the cell culture and purified by isolating the BMP-17,BMP-18 or related proteins from other proteinaceous materials with whichthey are co-produced as well as from other contaminants. The purifiedprotein may be assayed in accordance with the rat bone formation assaydescribed in Example 3.

[0101] Purification is carried out using standard techniques known tothose skilled in the art.

[0102] Protein analysis is conducted using standard techniques such asSDS-PAGE acrylamide [Laemmli, Nature 227:680 (1970)] stained with silver[Oakley, et al. Anal. Biochem. 105:361 (1980)] and by immunoblot[Towbin, et al. Proc. Natl. Acad. Sci. USA 76:4350 (1979)]

Example 6

[0103] Using Northern analysis, BMP-17, BMP-18 and related proteins canbe tested for their effects on various cell lines. Suitable cell linesinclude cell lines derived from E13 mouse limb buds. After 10 days oftreatment with BMP-17, BMP-18 or related protein, the cell phenotype isexamined histologically for indications of tissue differentiation. Inaddition, Northern analysis of mRNA from BMP-17, BMP-18 or relatedprotein treated cells can be performed for various markers including oneor more of the following markers for bone, cartilage and/ortendon/ligament, as described in Table IV: TABLE IV Marker BoneCartilage Tendon/Ligament Osteocalcin + − − Alkaline Phosphatase + − −Proteoglycan Core Protein +/−¹ +  +² Collagen Type I + + + Collagen TypeII +/−¹ +  +² Decorin + + + Elastin +/−³ ? +

Example 7

[0104] Embryonic Stem Cell Assay

[0105] In order to assay the effects of the BMP-17 and BMP-18 proteinsof the present invention, it is possible to assay the growth anddifferentiation effects in vitro on a number of available embryonic stemcell lines. One such cell line is ES-E14TG2, which is available from theAmerican Type Culture Collection in Rockville, Md.

[0106] In order to conduct the assay, cells may be propagated in thepresence of 100 units of LIF to keep them in an undifferentiated state.Assays are setup by first removing the LIF and aggregating the cells insuspension, in what is known as embryoid bodies. After 3 days theembryoid bodies are plated on gelatin coated plates (12 well plates forPCR analysis, 24 well plates for immunocytochemistry) and treated withthe proteins to be assayed. Cells are supplied with nutrients andtreated with the protein factor every 2-3 days. Cells may be adapted sothat assays may be conducted in media supplemented with 15% Fetal BovineSerum (FBS) or with CDM defined media containing much lower amounts ofFBS.

[0107] At the end of the treatment period (ranging from 7-21 days) RNAis harvested from the cells and analyzed by quantitative multiplex PCRfor the following markers: Brachyury, a mesodermal marker, AP-2, anectodermal marker, and HNF-3α an endodermal marker. Throughimmunocytochemistry, it is also possible to detect the differentiationof neuronal cells (glia and neurons), muscle cells (cardiomyocytes,skeletal and smooth muscle), and various other phenotype markers such asproteoglycan core protein (cartilage), and cytokeratins (epidermis).Since these cells have a tendency to differentiate autonomously when LIFis removed, the results are always quantitated by comparison to anuntreated control.

[0108] The foregoing descriptions detail presently preferred embodimentsof the present invention. Numerous modifications and variations inpractice thereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

1 7 1101 base pairs nucleic acid single linear cDNA pro_peptide 1..426mat_peptide 427..1098 CDS 1..1098 1 ATG CAG CCC CTG TGG CTC TGC TGG GCACTC TGG GTG TTG CCC CTG GCC 48 Met Gln Pro Leu Trp Leu Cys Trp Ala LeuTrp Val Leu Pro Leu Ala -142 -140 -135 -130 AGC CCC GGG GCC GCC CTG ACCGGG GAG CAG CTC CTG GGC AGC CTG CTG 96 Ser Pro Gly Ala Ala Leu Thr GlyGlu Gln Leu Leu Gly Ser Leu Leu -125 -120 -115 CGG CAG CTG CAG CTC AAAGAG GTG CCC ACC CTG GAC AGG GCC GAC ATG 144 Arg Gln Leu Gln Leu Lys GluVal Pro Thr Leu Asp Arg Ala Asp Met -110 -105 -100 -95 GAG GAG CTG GTCATC CCC ACC CAC GTG AGG GCC CAG TAC GTG GCC CTG 192 Glu Glu Leu Val IlePro Thr His Val Arg Ala Gln Tyr Val Ala Leu -90 -85 -80 CTG CAG CGC AGCCAC GGG GAC CGC TCC CGC GGA AAG AGG TTC AGC CAG 240 Leu Gln Arg Ser HisGly Asp Arg Ser Arg Gly Lys Arg Phe Ser Gln -75 -70 -65 AGC TTC CGA GAGGTG GCC GGC AGG TTC CTG GCG TTG GAG GCC AGC ACA 288 Ser Phe Arg Glu ValAla Gly Arg Phe Leu Ala Leu Glu Ala Ser Thr -60 -55 -50 CAC CTG CTG GTGTTC GGC ATG GAG CAG CGG CTG CCG CCC AAC AGC GAG 336 His Leu Leu Val PheGly Met Glu Gln Arg Leu Pro Pro Asn Ser Glu -45 -40 -35 CTG GTG CAG GCCGTG CTG CGG CTC TTC CAG GAG CCG GTC CCC AAG GCC 384 Leu Val Gln Ala ValLeu Arg Leu Phe Gln Glu Pro Val Pro Lys Ala -30 -25 -20 -15 GCG CTG CACAGG CAC GGG CGG CTG TCC CCG CGC AGC GCC CGG GCC CGG 432 Ala Leu His ArgHis Gly Arg Leu Ser Pro Arg Ser Ala Arg Ala Arg -10 -5 1 GTG ACC GTC GAGTGG CTG CGC GTC CGC GAC GAC GGC TCC AAC CGC ACC 480 Val Thr Val Glu TrpLeu Arg Val Arg Asp Asp Gly Ser Asn Arg Thr 5 10 15 TCC CTC ATC GAC TCCAGG CTG GTG TCC GTC CAC GAG AGC GGC TGG AAG 528 Ser Leu Ile Asp Ser ArgLeu Val Ser Val His Glu Ser Gly Trp Lys 20 25 30 GCC TTC GAC GTG ACC GAGGCC GTG AAC TTC TGG CAG CAG CTG AGC CGG 576 Ala Phe Asp Val Thr Glu AlaVal Asn Phe Trp Gln Gln Leu Ser Arg 35 40 45 50 CCC CGG CAG CCG CTG CTGCTA CAG GTG TCG GTG CAG AGG GAG CAT CTG 624 Pro Arg Gln Pro Leu Leu LeuGln Val Ser Val Gln Arg Glu His Leu 55 60 65 GGC CCG CTG GCG TCC GGC GCCCAC AAG CTG GTC CGC TTT GCC TCG CAG 672 Gly Pro Leu Ala Ser Gly Ala HisLys Leu Val Arg Phe Ala Ser Gln 70 75 80 GGG GCG CCA GCC GGG CTT GGG GAGCCC CAG CTG GAG CTG CAC ACC CTG 720 Gly Ala Pro Ala Gly Leu Gly Glu ProGln Leu Glu Leu His Thr Leu 85 90 95 GAC CTT GGG GAC TAT GGA GCT CAG GGCGAC TGT GAC CCT GAA GCA CCA 768 Asp Leu Gly Asp Tyr Gly Ala Gln Gly AspCys Asp Pro Glu Ala Pro 100 105 110 ATG ACC GAG GGC ACC CGC TGC TGC CGCCAG GAG ATG TAC ATT GAC CTG 816 Met Thr Glu Gly Thr Arg Cys Cys Arg GlnGlu Met Tyr Ile Asp Leu 115 120 125 130 CAG GGG ATG AAG TGG GCC GAG AACTGG GTG CTG GAG CCC CCG GGC TTC 864 Gln Gly Met Lys Trp Ala Glu Asn TrpVal Leu Glu Pro Pro Gly Phe 135 140 145 CTG GCT TAT GAG TGT GTG GGC ACCTGC CGG CAG CCC CCG GAG GCC CTG 912 Leu Ala Tyr Glu Cys Val Gly Thr CysArg Gln Pro Pro Glu Ala Leu 150 155 160 GCC TTC AAG TGG CCG TTT CTG GGGCCT CGA CAG TGC ATC GCC TCG GAG 960 Ala Phe Lys Trp Pro Phe Leu Gly ProArg Gln Cys Ile Ala Ser Glu 165 170 175 ACT GCC TCG CTG CCC ATG ATC GTCAGC ATC AAG GAG GGA GGC AGG ACC 1008 Thr Ala Ser Leu Pro Met Ile Val SerIle Lys Glu Gly Gly Arg Thr 180 185 190 AGG CCC CAG GTG GTC AGC CTG CCCAAC ATG AGG GTG CAG AAG TGC AGC 1056 Arg Pro Gln Val Val Ser Leu Pro AsnMet Arg Val Gln Lys Cys Ser 195 200 205 210 TGT GCC TCG GAT GGT GCG CTCGTG CCA AGG AGG CTC CAG CCA 1098 Cys Ala Ser Asp Gly Ala Leu Val Pro ArgArg Leu Gln Pro 215 220 TAG 1101 366 amino acids amino acid linearprotein 2 Met Gln Pro Leu Trp Leu Cys Trp Ala Leu Trp Val Leu Pro LeuAla -142 -140 -135 -130 Ser Pro Gly Ala Ala Leu Thr Gly Glu Gln Leu LeuGly Ser Leu Leu -125 -120 -115 Arg Gln Leu Gln Leu Lys Glu Val Pro ThrLeu Asp Arg Ala Asp Met -110 -105 -100 -95 Glu Glu Leu Val Ile Pro ThrHis Val Arg Ala Gln Tyr Val Ala Leu -90 -85 -80 Leu Gln Arg Ser His GlyAsp Arg Ser Arg Gly Lys Arg Phe Ser Gln -75 -70 -65 Ser Phe Arg Glu ValAla Gly Arg Phe Leu Ala Leu Glu Ala Ser Thr -60 -55 -50 His Leu Leu ValPhe Gly Met Glu Gln Arg Leu Pro Pro Asn Ser Glu -45 -40 -35 Leu Val GlnAla Val Leu Arg Leu Phe Gln Glu Pro Val Pro Lys Ala -30 -25 -20 -15 AlaLeu His Arg His Gly Arg Leu Ser Pro Arg Ser Ala Arg Ala Arg -10 -5 1 ValThr Val Glu Trp Leu Arg Val Arg Asp Asp Gly Ser Asn Arg Thr 5 10 15 SerLeu Ile Asp Ser Arg Leu Val Ser Val His Glu Ser Gly Trp Lys 20 25 30 AlaPhe Asp Val Thr Glu Ala Val Asn Phe Trp Gln Gln Leu Ser Arg 35 40 45 50Pro Arg Gln Pro Leu Leu Leu Gln Val Ser Val Gln Arg Glu His Leu 55 60 65Gly Pro Leu Ala Ser Gly Ala His Lys Leu Val Arg Phe Ala Ser Gln 70 75 80Gly Ala Pro Ala Gly Leu Gly Glu Pro Gln Leu Glu Leu His Thr Leu 85 90 95Asp Leu Gly Asp Tyr Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala Pro 100 105110 Met Thr Glu Gly Thr Arg Cys Cys Arg Gln Glu Met Tyr Ile Asp Leu 115120 125 130 Gln Gly Met Lys Trp Ala Glu Asn Trp Val Leu Glu Pro Pro GlyPhe 135 140 145 Leu Ala Tyr Glu Cys Val Gly Thr Cys Arg Gln Pro Pro GluAla Leu 150 155 160 Ala Phe Lys Trp Pro Phe Leu Gly Pro Arg Gln Cys IleAla Ser Glu 165 170 175 Thr Ala Ser Leu Pro Met Ile Val Ser Ile Lys GluGly Gly Arg Thr 180 185 190 Arg Pro Gln Val Val Ser Leu Pro Asn Met ArgVal Gln Lys Cys Ser 195 200 205 210 Cys Ala Ser Asp Gly Ala Leu Val ProArg Arg Leu Gln Pro 215 220 1101 base pairs nucleic acid single linearDNA (genomic) pro_peptide 1..405 mat_peptide 406..1098 CDS 1..1098 3 ATGTGG CCC CTG TGG CTC TGC TGG GCA CTC TGG GTG CTG CCC CTG GCT 48 Met TrpPro Leu Trp Leu Cys Trp Ala Leu Trp Val Leu Pro Leu Ala -135 -130 -125-120 GGC CCC GGG GCG GCC CTG ACC GAG GAG CAG CTC CTG GGC AGC CTG CTG 96Gly Pro Gly Ala Ala Leu Thr Glu Glu Gln Leu Leu Gly Ser Leu Leu -115-110 -105 CGG CAG CTG CAG CTC AGC GAG GTG CCC GTA CTG GAC AGG GCC GACATG 144 Arg Gln Leu Gln Leu Ser Glu Val Pro Val Leu Asp Arg Ala Asp Met-100 -95 -90 GAG AAG CTG GTC ATC CCC GCC CAC GTG AGG GCC CAG TAT GTA GTCCTG 192 Glu Lys Leu Val Ile Pro Ala His Val Arg Ala Gln Tyr Val Val Leu-85 -80 -75 CTG CGG CGC AGC CAC GGG GAC CGC TCC CGC GGA AAG AGG TTC AGCCAG 240 Leu Arg Arg Ser His Gly Asp Arg Ser Arg Gly Lys Arg Phe Ser Gln-70 -65 -60 AGC TTC CGA GAG GTG GCC GGC AGG TTC CTG GCG TCG GAG GCC AGCACA 288 Ser Phe Arg Glu Val Ala Gly Arg Phe Leu Ala Ser Glu Ala Ser Thr-55 -50 -45 -40 CAC CTG CTG GTG TTC GGC ATG GAG CAG CGG CTG CCG CCC AACAGC GAG 336 His Leu Leu Val Phe Gly Met Glu Gln Arg Leu Pro Pro Asn SerGlu -35 -30 -25 CTG GTG CAG GCC GTG CTG CGG CTC TTC CAG GAG CCG GTC CCCAAG GCC 384 Leu Val Gln Ala Val Leu Arg Leu Phe Gln Glu Pro Val Pro LysAla -20 -15 -10 GCG CTG CAC AGG CAC GGG CGG CTG TCC CCG CGC AGC GCC CAGGCC CGG 432 Ala Leu His Arg His Gly Arg Leu Ser Pro Arg Ser Ala Gln AlaArg -5 1 5 GTG ACC GTC GAG TGG CTG CGC GTC CGC GAC GAC GGC TCC AAC CGCACC 480 Val Thr Val Glu Trp Leu Arg Val Arg Asp Asp Gly Ser Asn Arg Thr10 15 20 25 TCC CTC ATC GAC TCC AGG CTG GTG TCC GTC CAC GAG AGC GGC TGGAAG 528 Ser Leu Ile Asp Ser Arg Leu Val Ser Val His Glu Ser Gly Trp Lys30 35 40 GCC TTC GAC GTG ACC GAG GCC GTG AAC TTC TGG CAG CAG CTG AGC CGG576 Ala Phe Asp Val Thr Glu Ala Val Asn Phe Trp Gln Gln Leu Ser Arg 4550 55 CCC CGG CAG CCG CTG CTG CTA CAG GTG TCG GTG CAG AGG GAG CAT CTG624 Pro Arg Gln Pro Leu Leu Leu Gln Val Ser Val Gln Arg Glu His Leu 6065 70 GGC CCG CTG GCG TCC GGC GCC CAC AAG CTG GTC CGC TTT GCC TCG CAG672 Gly Pro Leu Ala Ser Gly Ala His Lys Leu Val Arg Phe Ala Ser Gln 7580 85 GGG GCG CCA GCC GGG CTT GGG GAG CCC CAG CTG GAG CTG CAC ACC CTG720 Gly Ala Pro Ala Gly Leu Gly Glu Pro Gln Leu Glu Leu His Thr Leu 9095 100 105 GAC CTC AGG GAC TAT GGA GCT CAG GGC GAC TGT GAC CCT GAA GCACCA 768 Asp Leu Arg Asp Tyr Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala Pro110 115 120 ATG ACC GAG GGC ACC CGC TGC TGC CGC CAG GAG ATG TAC ATT GACCTG 816 Met Thr Glu Gly Thr Arg Cys Cys Arg Gln Glu Met Tyr Ile Asp Leu125 130 135 CAG GGG ATG AAG TGG GCC AAG AAC TGG GTG CTG GAG CCC CCG GGCTTC 864 Gln Gly Met Lys Trp Ala Lys Asn Trp Val Leu Glu Pro Pro Gly Phe140 145 150 CTG GCT TAC GAG TGT GTG GGC ACC TGC CAG CAG CCC CCG GAG GCCCTG 912 Leu Ala Tyr Glu Cys Val Gly Thr Cys Gln Gln Pro Pro Glu Ala Leu155 160 165 GCC TTC AAT TGG CCA TTT CTG GGG CCG CGA CAG TGT ATC GCC TCGGAG 960 Ala Phe Asn Trp Pro Phe Leu Gly Pro Arg Gln Cys Ile Ala Ser Glu170 175 180 185 ACT GCC TCG CTG CCC ATG ATC GTC AGC ATC AAG GAG GGA GGCAGG ACC 1008 Thr Ala Ser Leu Pro Met Ile Val Ser Ile Lys Glu Gly Gly ArgThr 190 195 200 AGG CCC CAG GTG GTC AGC CTG CCC AAC ATG AGG GTG CAG AAGTGC AGC 1056 Arg Pro Gln Val Val Ser Leu Pro Asn Met Arg Val Gln Lys CysSer 205 210 215 TGT GCC TCG GAT GGG GCG CTC GTG CCA AGG AGG CTC CAG CCA1098 Cys Ala Ser Asp Gly Ala Leu Val Pro Arg Arg Leu Gln Pro 220 225 230TAG 1101 366 amino acids amino acid linear protein 4 Met Trp Pro Leu TrpLeu Cys Trp Ala Leu Trp Val Leu Pro Leu Ala -135 -130 -125 -120 Gly ProGly Ala Ala Leu Thr Glu Glu Gln Leu Leu Gly Ser Leu Leu -115 -110 -105Arg Gln Leu Gln Leu Ser Glu Val Pro Val Leu Asp Arg Ala Asp Met -100 -95-90 Glu Lys Leu Val Ile Pro Ala His Val Arg Ala Gln Tyr Val Val Leu -85-80 -75 Leu Arg Arg Ser His Gly Asp Arg Ser Arg Gly Lys Arg Phe Ser Gln-70 -65 -60 Ser Phe Arg Glu Val Ala Gly Arg Phe Leu Ala Ser Glu Ala SerThr -55 -50 -45 -40 His Leu Leu Val Phe Gly Met Glu Gln Arg Leu Pro ProAsn Ser Glu -35 -30 -25 Leu Val Gln Ala Val Leu Arg Leu Phe Gln Glu ProVal Pro Lys Ala -20 -15 -10 Ala Leu His Arg His Gly Arg Leu Ser Pro ArgSer Ala Gln Ala Arg -5 1 5 Val Thr Val Glu Trp Leu Arg Val Arg Asp AspGly Ser Asn Arg Thr 10 15 20 25 Ser Leu Ile Asp Ser Arg Leu Val Ser ValHis Glu Ser Gly Trp Lys 30 35 40 Ala Phe Asp Val Thr Glu Ala Val Asn PheTrp Gln Gln Leu Ser Arg 45 50 55 Pro Arg Gln Pro Leu Leu Leu Gln Val SerVal Gln Arg Glu His Leu 60 65 70 Gly Pro Leu Ala Ser Gly Ala His Lys LeuVal Arg Phe Ala Ser Gln 75 80 85 Gly Ala Pro Ala Gly Leu Gly Glu Pro GlnLeu Glu Leu His Thr Leu 90 95 100 105 Asp Leu Arg Asp Tyr Gly Ala GlnGly Asp Cys Asp Pro Glu Ala Pro 110 115 120 Met Thr Glu Gly Thr Arg CysCys Arg Gln Glu Met Tyr Ile Asp Leu 125 130 135 Gln Gly Met Lys Trp AlaLys Asn Trp Val Leu Glu Pro Pro Gly Phe 140 145 150 Leu Ala Tyr Glu CysVal Gly Thr Cys Gln Gln Pro Pro Glu Ala Leu 155 160 165 Ala Phe Asn TrpPro Phe Leu Gly Pro Arg Gln Cys Ile Ala Ser Glu 170 175 180 185 Thr AlaSer Leu Pro Met Ile Val Ser Ile Lys Glu Gly Gly Arg Thr 190 195 200 ArgPro Gln Val Val Ser Leu Pro Asn Met Arg Val Gln Lys Cys Ser 205 210 215Cys Ala Ser Asp Gly Ala Leu Val Pro Arg Arg Leu Gln Pro 220 225 230 15base pairs nucleic acid single linear DNA (genomic) 5 CATGGGCAGC TCGAG15 34 base pairs nucleic acid single linear DNA (genomic) 6 CTGCAGGCGAGCCTGAATTC CTCGAGCCAT CATG 34 68 base pairs nucleic acid single linearDNA (genomic) 7 CGAGGTTAAA AAACGTCTAG GCCCCCCGAA CCACGGGGAC GTGGTTTTCCTTTGAAAAAC 60 ACGATTGC 68

What is claimed is:
 1. An isolated DNA molecule comprising a DNAsequence selected from the group consisting of: (a) nucleotides #1, 232,406, 427, 751 or 796 to #1059 or 1098 of SEQ ID NO:1; (b) nucleotidesencoding amino acids #-142, -65, -7, 1, 109 or 124 to #211 or 224 of SEQID NO:2; (c) nucleotides #1, 232, 406, 427, 751 or 796 to #1059 or 1098of SEQ ID NO:3; (d) nucleotides encoding amino acids #-135, -58, 1, 8,116 or 131 to #218 or 231 of SEQ ID NO:4; and (e) naturally occurringhuman allelic sequences and equivalent degenerative codon sequences of(a), (b), (c) or (d).
 2. A host cell transformed with the DNA sequenceof claim
 1. 3. A vector comprising a DNA molecule of claim 1 inoperative association with an expression control sequence therefor.
 4. Ahost cell transformed with the vector of claim
 3. 5. An isolated DNAmolecule comprising a DNA sequence consisting of nucleotides #427 to#1098 of SEQ ID NO:1.
 6. An isolated DNA molecule comprising a DNAsequence consisting of nucleotides #406 to #1098 of SEQ ID NO:3.
 7. Avector comprising a DNA molecule of claim 5 in operative associationwith an expression control sequence therefor.
 8. A host cell transformedwith the vector of claim
 7. 9. A vector comprising a DNA molecule ofclaim 6 in operative association with an expression control sequencetherefor.
 10. A host cell transformed with the vector of claim
 9. 11. Amethod for producing a purified human bone morphogenetic protein-17(BMP) and BMP-18 protein, said method comprising the steps of: (a)culturing a host cell transformed with a DNA molecule according to claim1; and (b) recovering and purifying said human BMP-17 and BMP-18 proteinfrom the culture medium.
 12. The method of claim 11, wherein said hostcell is transformed with a DNA molecule comprising a DNA coding sequenceconsisting of nucleotide #427 to #1098 of SEQ ID NO:
 1. 13. The methodof claim 12, wherein said host cell is a mammalian cell and the DNAmolecule further comprises a DNA sequence encoding a propeptide from amember of the TGF-β superfamily of proteins, said DNA sequence encodinga propeptide being linked in proper reading frame to the DNA codingsequence.
 14. The method of claim 11, wherein said host cell istransformed with a DNA molecule comprising a DNA coding sequenceconsisting of nucleotide #406 to #1098 of SEQ ID NO:
 3. 15. The methodof claim 14, wherein said host cell is a mammalian cell and the DNAmolecule further comprises a DNA sequence encoding a propeptide from amember of the TGF-β superfamily of proteins, said DNA sequence encodinga propeptide being linked in proper reading frame to the DNA codingsequence.
 16. A purified bone morphogenetic protein-17 polypeptidecomprising the amino acid sequence from amino acid #1 to #224 as setforth in SEQ ID NO:
 2. 17. A purified BMP-17 polypeptide of claim 16wherein said polypeptide is a dimer and wherein at least one subunitcomprises the amino acid sequence from amino acid #1 to #224 of SEQ IDNO:
 2. 18. A purified bone morphogenetic protein-17 polypeptide producedby the steps of: (a) culturing a cell transformed with a DNA moleculeaccording to claim 5; and (b) recovering and purifying from said culturemedium a polypeptide comprising the amino acid sequence from amino acid#1 to amino acid #224 of SEQ ID NO:
 2. 19. A purified bone morphogeneticprotein-17 polypeptide according to claim 18, wherein said polypeptideis a dimer comprising two subunits, wherein one subunit comprises theamino acid sequence from amino acid #1 to amino acid #224 of SEQ ID NO:2, and one subunit comprises an amino acid sequence for a bonemorphogenetic protein selected from the group consisting of BMP-1,BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9 BMP-10, BMP-11,BMP-12, BMP-13, BMP-15 and BMP-16.
 20. A purified bone morphogeneticprotein-18 polypeptide comprising the amino acid sequence from aminoacid #1 to #231 as set forth in SEQ ID NO:
 4. 21. A purified BMP-18polypeptide of claim 20 wherein said polypeptide is a dimer and whereinat least one subunit comprises the amino acid sequence from amino acid#1 to#231 of SEQ ID NO:
 4. 22. A purified bone morphogenetic protein-18polypeptide produced by the steps of: (a) culturing a cell transformedwith a DNA molecule according to claim 6; and (b) recovering andpurifying from said culture medium a polypeptide comprising the aminoacid sequence from amino acid #1 to amino acid #231 of SEQ ID NO:
 4. 23.A purified bone morphogenetic protein-18 polypeptide according to claim22, wherein said polypeptide is a dimer comprising two subunits, whereinone subunit comprises the amino acid sequence from amino acid #1 toamino acid #231 of SEQ ID NO: 4, and one subunit comprises an amino acidsequence for a bone morphogenetic protein selected from the groupconsisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8,BMP-9 BMP-10, BMP-11, BMP-12, BMP-13, BMP-15 and BMP-16.
 24. A chimericDNA molecule comprising a DNA sequence encoding a propeptide from amember of the TGF-β superfamily of proteins linked in frame to a DNAsequence encoding a bone morphogenetic protein-17 polypeptide, saidBMP-17 polypeptide comprising amino acid #1 to #224 of SEQ ID NO:
 2. 25.A chimeric DNA molecule comprising a DNA sequence encoding a propeptidefrom a member of the TGF-β superfamily of proteins linked in frame to aDNA sequence encoding a bone morphogenetic protein-18 polypeptide, saidBMP-18 polypeptide comprising amino acid #1 to #231 of SEQ ID NO:
 4. 26.Antibodies to a purified BMP-17 protein according to claim
 16. 27.Antibodies to a purified BMP-18 protein according to claim 20.